Transcriptomic analysis of blood-fed female mosquitoes

Females were fed with human blood, and 15 guts were dissected into TRIzol after 6 hours, after 20 hours, and from unfed females. After homogenization with 2.8-mm ceramic beads (CK28R, Precellys), RNA was extracted with the Direct-zol RNA Mini-prep Kit (Zymo Research) including on-column DNase treatment. Four biological replicates per condition were subjected to RNA sequencing (RNA-seq). Libraries were prepared with the NEB Next Ultra RNA Library Prep Kit and sequenced on a NovaSeq 6000 Illumina platform (instrument HWI-ST1276) generating 150-bp paired-end reads (GenBank accession PRJNA822650). Replicate 1 for MM-CP without blood meal (MMCP_N_1) was identified as outlier with squared Pearson correlation coefficients with the other three biological replicates below 0.84 and hence removed from further analysis. Sequencing reads were mapped to the A. gambiae PEST genome (AgamP4.13, GCA_000005575.2 supplemented with the MM-CP construct reference) using HISAT2 software v2.0.5 (with parameters --dta --phred33) (54 (link)). Differential expression was assessed with DESeq2 v1.20.0. GO enrichment analysis was performed using TopGO (55 ) with a pruning factor of 50 using a P value cutoff of P = 0.01.